It is well known in the art that DNA as well as RNA can be methylated. The base 5-methylcytosine is the most frequent covalently modified base found in the DNA of eukaryotic cells. DNA methylation plays an important biological role in, for example, regulating transcription, genomic imprinting, and tumorigenesis (for review see, e.g., Millar et al.: Five not four: History and significance of the fifth base; in The Epigenome, S. Beck and A. Olek (eds.), Wiley-VCH Publishers, Weinheim 2003, pp. 3-20). The identification of 5-methylcytosine is of particular interest in the area of cancer diagnosis. But the identification of methylation is difficult. Cytosine and 5-methylcytosine have the same base-pairing behavior, making 5-methylcytosine difficult to detect using particular standard methods. The conventional DNA analysis methods based on hybridization, for example, are not applicable. In addition, the methylation information is lost completely by the amplification by means of PCR.
Accordingly, current methods for DNA methylation analysis are based on at least two different approaches. The first approach utilizes methylation specific restriction enzymes to distinguish methylated DNA, based on methylation specific DNA cleavage. The second approach comprises selective chemical conversion (e.g., bisulfite treatment; see e.g. WO 2005/038051) of unmethylated cytosines to uracil while methylated cytosines remain unchanged. Uracil has the same base pairing behavior as thymine. It therefore forms base pairs with adenine. Instead, 5-methylcytosine hybridizes with guanine still after bisulfite treatment. It is therewith possible to differentiate between methylated and unmethylated cytosines. Alternatively, cytosine may be converted by enzymes like for example cytidine-deaminase which converts unmethylated cytosine faster as methylated cytosine. An appropriate enzyme is described by Bransteitter et al. (Bransteitter et al.: “Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of Rnase”. PNAS 2003, 100(7): 4102-4107; WO 2005/005660).
The enzymatically or chemically pretreated DNA generated in these approaches is typically sequenced and/or pre-amplified and analyzed in different ways (see, e.g., WO 02/072880 pp. 1 ff; Fraga and Estella: DNA methylation: a profile of methods and applications; Biotechniques, 33:632, 634, 636-49, 2002). A wide range of methods exists to detect genomic DNA methylation, including approaches to detect genome-wide and gene-specific methylation levels. Some embodiments of these methods allow also a quantification of methylation i.e. the determination of the amount of DNA molecules that are methylated or unmethylated at said pre-defined CpG position within a mixture of DNA molecules.
Various methylation assay procedures are known in the art, and can be used in conjunction with the present invention. Most methods used to analyze the methylation state or level of a specific sequence are based on bisulfite modification of the DNA or other reagents with the same treatment behavior as bisulfite. Following the treatment, the methylation status can be assessed as a sequence difference for example by direct sequencing, Pyrosequencing, Next Generation Sequencing, primer extension, COBRA, MLA, MSP, MethyLight, HM, QM, and HQM.